The present invention relates to a cytokeratin 8/18 complex-specific autoantibody or a fragment comprising an antigen-binding site (paratope) thereof, use thereof in the diagnosis of breast cancer, a polypeptide having an amino acid sequence of an epitope specifically binding to the autoantibody, a composition for diagnosing breast cancer comprising an agent capable of measuring an expression level of the autoantibody or the fragment comprising an antigen-binding site thereof, a hybridoma cell line producing the autoantibody, and a kit for diagnosing breast cancer comprising the composition of the present invention. Further, the present invention relates to a method for diagnosing breast cancer, comprising the step of detecting the cytokeratin 8/18 complex-specific autoantibody or the fragment comprising the antigen-binding site thereof using the composition of the present invention, and a method for screening a therapeutic agent for breast cancer using the autoantibody.
Breast cancer has the highest incidence rates among various cancers in most OECD countries. In Korea, the incidence has also been increasing, concurrent with rising prevalence of obesity and the Westernization of dietary habits. Diagnosis of breast cancer is usually made by mammography, ultrasonography, and Magnetic Resonance imaging (MRI). Mammography is an X-ray examination of the compressed breast, and is useful for detection of small breast cancers. However, many Korean women have less fatty tissue and denser fibrous tissue, and thus it is difficult to detect calcification as an early warning sign of breast cancer. Therefore, a mammogram is usually performed together with ultrasonography which is useful for the detection of solid and cystic breast masses. These mechanical examinations are conducted by a simple examination procedure, but have limited detection accuracy because they rely on a reader's subjective interpretation. Therefore, subsequent tissue biopsy is required. Such tissue biopsy is an invasive method including examination of breast cancer-specific protein expression in the tissue or tissue observation, and requires considerable costs while imposing emotional, physical, and economic burdens on patients. In order to overcome these drawbacks, there is a need to develop an easy and simple diagnostic method for breast cancer.
Meanwhile, the immune system is constructed as a peculiar system distinguishing the self from non-self at an early stage of development, and develops to induce antigen-antibody reaction (humoral immune response) and cellular immune response against foreign antigens only which are exposed to the immune system under normal conditions. However, the generation of antibodies against self-antigens was observed in specific diseases, which is attributed to extracellular release of intracellular antigens due to abnormal expression site of the corresponding antigens, modification of forms, or other aberrant characteristics. Since the 1970s, it has been also reported that the generation of autoantibodies against cancer cell-derived antigens is observed in carcinogenesis, accompanied by abnormal tumor growth, and they are called tumor-associated antigens and tumor-associated autoantibodies. Autoantibodies generated by tumor-associated antigens pre-exist and are found before the onset of disease, suggesting the availability as a biomarker for the early diagnosis.
Until now, various tumor-associated antigens have been identified. Among them, overexpression of HER-2/neu oncoprotein found in 20-30% of breast cancer patients is known to induce autoantibodies, and a tumor suppressor protein p53 is also reported to induce autoantibodies. The cell proliferation-associated proteins, cyclin B1 and CENP-F (centromere protein F) are also known to induce autoantibodies. These results suggest the presence of autoantibodies against a larger number of tumor-associated antigens, and many trials have been made to screen tumor-associated autoantibodies that can be utilized as a cancer biomarker.
For identification of tumor-associated autoantibodies, SEREX (selological analysis of recombinant cDNA expression libraries of human tumors with autologous serum), which is a conventional method of searching autoantibodies by analyzing reactivity of cancer patient sera with cancer cell-derived gene expression libraries, has been used. However, it is difficult to express cancer cell-derived proteins with maximal diversity at the library level. In addition, even after transcription, final protein products undergo posttranslational modification (PTM) such as phosphorylation and glycosylation, which is not reflected in the protein expression libraries. Therefore, the method is not sufficient to detect autoantigens. Alternatively, proteomics-based identification of autoantibodies has been recently conducted. The proteomics-based method includes the procedures of performing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of cancer cell-derived proteins, detecting protein spots showing reactivity using cancer patient serum as an autoantibody sample, and identifying the proteins by mass spectrometry, and is commonly abbreviated to SERPA (serological proteome analysis). MAPPING (multiple affinity protein profiling) is also used, in which an affinity chromatography resin is conjugated with antibodies isolated from the patient blood, and cancer cell-derived proteins are applied thereto to identify binding proteins by mass; spectrometry. In addition, cancer cell lysates are separated into several thousand individual fractions to fabricate a protein chip, and the reactivity of patient blood thereto is examined to identify autoantibodies. Such various proteomics-based identification methods are advantageous in that the reactivity of antibodies against cancer cell-derived proteins with posttranslational modifications (PTM) can be directly examined, and thus are able to identify autoantibodies that cannot be identified by SEREX.
However, the above methods still include other limitations. First, there is a problem in the quantity of antibodies. When an analyte is a mixture of two or more components, a relatively large amount of the analyte takes precedence over other analyte in the analysis. This means that minorities are possibly excluded from the analyzable range. In this context, a patient's serum is a mixture of numerous antibodies, an analytically measurable range is determined by the differences in quantity of the constituting autoantibodies and their affinity for antigens, and therefore analysis on the desired autoantibodies could be impractical. Second, due to the dependence of the autoantibody samples to be analyzed on a patient, there is a limitation in the systematic analysis of autoantibody production according to carcinogenesis, and there is also difficulty in collecting a large enough amount of blood samples from patients, which becomes an obstacle to further studies. Finally, there is a problem in the preservation of epitopes reacting with antibodies. According to the current immunological knowledge, there are two types of epitopes, sequential epitopes and conformational epitopes. In vivo induction of antigen-specific antibody production reflects an antigen being encountered by the immune cell antibody for the first time, which means that the antigen-antibody reaction occurs in a solution, and the antigen protein has a structure of maintaining its solubilized form in the blood. Thus, ex vivo test of antigen-antibody reaction is preferably performed in the solution, because their natural binding pattern is well reflected in the solution. In the above mentioned SERPA, however, two-dimensional electrophoresis is performed for the analysis of a protein mixture. That is, proteins to be analyzed are denatured by using SDS and urea, and then their reaction with antibodies is examined. Thus, if the epitope is a sequential epitope, it is detectable, but if the epitope is a conformational epitope, not detectable.
Although the previous studies on autoantibodies suggested their availability as a carcinogenesis-associated maker, their diagnostic effects are not satisfactory, and there is still a limitation in the availability of autoantibodies as a biomarkers for cancer diagnosis. Further, the autoantibody detection methods have limitations in practical application because they do not include a large number of cases or do require excessive experiments. Actually, there are still difficulties in the identification of autoantibodies as a diagnostic marker for cancer.